Endogenously elevated bilirubin modulates kidney function and protects from circulating oxidative stress in a rat model of adenine-induced kidney failure

Mildly elevated bilirubin is associated with a reduction in the presence and progression of chronic kidney disease and related mortality, which may be attributed to bilirubin’s antioxidant properties. This study investigated whether endogenously elevated bilirubin would protect against adenine-induced kidney damage in male hyperbilirubinaemic Gunn rats and littermate controls. Animals were orally administered adenine or methylcellulose solvent (vehicle) daily for 10 days and were then monitored for 28 days. Serum and urine were assessed throughout the protocol for parameters of kidney function and antioxidant/oxidative stress status and kidneys were harvested for histological examination upon completion of the study. Adenine-treated animals experienced weight-loss, polyuria and polydipsia; however, these effects were significantly attenuated in adenine-treated Gunn rats. No difference in the presence of dihydroadenine crystals, lymphocytic infiltration and fibrosis were noted in Gunn rat kidneys versus controls. However, plasma protein carbonyl and F2-isoprostane concentrations were significantly decreased in Gunn rats versus controls, with no change in urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine or kidney tissue F2-isoprostane concentrations. These data indicated that endogenously elevated bilirubin specifically protects from systemic oxidative stress in the vascular compartment. These data may help to clarify the protective relationship between bilirubin, kidney function and cardiovascular mortality in clinical investigations.

Serum and urinary parameters of kidney function. Serum and urine biochemistries of adenine-administered (Table S1) and vehicle-control animals (Table S2) were examined at baseline and throughout the observation period of 28 days. No significant (P < 0.05) differences in albumin, total protein or glucose were noted between the groups. Gunn rats had reduced total cholesterol and greater triglyceride concentrations versus controls (Table S1). Serum liver enzyme activities were elevated in adenine-treated groups on day 0, with increased ALT and reduced GGT activities demonstrated in GA compared to WA at day 10. Serum urea and creatinine concentrations were mildly increased in Gunn rats compared to Wistar controls after adenine treatment for 10 days (i.e. day 0; Fig. 3a and b; P < 0.01). However, all concentrations returned to baseline values after 14-28 days. There were no changes in serum uric acid and calcium concentrations between adenine treated groups (Fig. 3c,d). A mild, yet significant (P < 0.01) increase in serum phosphate concentrations was also observed in GA at day 0 (Fig. 3e). Total urinary excretion of urea, creatinine and total protein were increased after adenine dosing at day 0 in all animals and generally continued to increase over the observation period of 28 days (Fig. 4a-c). Urea and creatinine output were significantly reduced in GA at day 14 and 28 (P < 0.05, Fig. 3a,b). Similarly, GA had significantly reduced total protein and phosphate excretion at day 14 and 28, respectively (Fig. 3c,e).
Urinary excretion of electrolytes progressively increased in animals after adenine-treatment at day 0 (Fig. 5). Adenine-treated Gunn rats experienced increased urinary excretion of sodium at day 0 (P < 0.05); however, was significantly reduced at day 14 when compared to controls (P < 0.05; Fig. 5c).
Antioxidant/oxidative stress status. Direct and total bilirubin concentrations were significantly elevated in Gunn rats versus controls throughout the protocol (Table S3 and S4). However, reduced Figure 1. Effects of adenine-induced tubulo-interstitial injury on body weight. (a) Percentage body weight loss or gain (from day − 10) in animals during adenine and methylcellulose treatment for 10 days, followed by 28 days of observation (∑, GA, n = 12; ■, WA, n = 12; , GC, n = 9; □ , WC, n = 6). Error bars have been removed to improve clarity of this graph. (b) Relative body weight loss of adenine treated animals (GA/WA) versus their methylcellulose controls (GC/WC) during adenine and methylcellulose treatment for 10 days, followed by 28 days of observation. Data are expressed as the mean ± standard deviation. *P > 0.05 between the adenine-treated groups. thiols, when expressed relative to protein, and reduced glutathione (a thiol containing antioxidant) were not significantly different between WA and GA at all-time points ( Fig. 6a; Table S3 and S4).
No difference in GSSG concentrations and the GSH:GSSG ratio were noted between the groups at any time-point (Table S3). Furthermore, plasma protein carbonyl concentrations were significantly (P < 0.05) decreased in GA compared to WA throughout the protocol (Fig. 7a). Plasma F 2 -isoprostane concentrations were also significantly (P < 0.01; Fig. 7c) decreased in GA compared to WA at day 28. No difference was observed in kidney F 2 -isoprostane content between the groups (Fig. 7d). Furthermore, urinary 8-oxo-7,8-dihydro-2′ -deoxyguanosine (8-oxodG) concentrations did not differ between the groups at any time-point (Fig. 7b).
Histopathology. There were no statistical differences in the distribution of histological scoring of kidneys after 28 days when compared the adenine-treated groups (Table S5). Dihydroadenine crystals were detected in the hematoxylin-eosin (HE) stained animal kidneys treated with adenine and were highlighted using polarised light (Fig. 8a,b). Representative slides revealed similar infiltration of foreign body giant cells reactive to the crystal in the kidney interstitium of animals treated with adenine ( Fig. 8b). Lymphocytic infiltration and interstitial calcification were detected via HE staining (Fig. 8a) and interstitial fibrosis was observed in Masson trichrome stained sections in adenine treated animals (Fig. 8c). The kidney morphology of animals treated with methylcellulose stained with HE appeared unremarkable ( Figure S2), the scoring of which is presented in Table S6.

Figure 2. Effects of adenine-induced tubulo-interstitial injury on urine output and water consumption.
Urine output (a) and water consumption (b) in animals treated with adenine for 10 days and then monitored for 28 days (∑, GA; ■, WA; n = 12 per group). Data are expressed as a mean ± standard deviation. *P < 0.05 between GA and WA groups.

Discussion
The main findings of this study were that hyperbilirubinaemic Gunn rats, when treated with adenine, experienced reduced polyuria and polydipsia and concomitantly regained and maintained their body mass compared to normo-bilirubinaemic animals. Although hyperbilirubinaemia did not improve systemic markers of kidney function, Gunn rats evidenced an improved circulating antioxidant status (total bilirubin) and decreased circulating oxidative stress biomarker concentrations (protein carbonyl and F 2 -isoprostanes) compared to controls after adenine administration. Decreased concentrations of circulating/systemic markers of oxidation were not accompanied by similar changes in markers that reflect cell-based oxidative stress (i.e. kidney F 2 -isoprostanes and urinary 8-oxodG) suggesting that bilirubin's protective effects were largely confined to the vascular compartment. This is the first study to demonstrate that bilirubin may protect circulating proteins and lipids in blood from adenine-induced oxidative stress in vivo, providing further insight into protection from vascular injury during CKD in individuals with elevated bilirubin concentrations (e.g. GS).
Adenine is metabolised to 2,8-dihydroxyadenine (DHA), which is not readily excreted by the kidney, resulting in the deposition of DHA crystals in kidney tubules. This deposition induces chronic kidney disease due to interstitial inflammation, tubular damage and fibrosis 25,27 . Adenine-induced chronic kidney failure in this rat model mimics the clinical condition of human chronic kidney failure 25,[27][28][29] and several studies have reported a reduction in body weight in adenine-fed animals 27,28 . Terai et al. administered adenine (100 mg/rat) and methylcellulose daily for 12 days 28 , which was accompanied by significant body mass reduction compared to solvent control treated animals. Body mass reduction upon adenine feeding is associated with reduced food consumption (approximately one half versus controls) 28 and azotemia as documented here. Body weight decreased similarly in adenine treated Gunn and Wistar animals in this study (10 days of adenine administration; 300 mg/kg/day). Compared to WA, GA regained greater body mass from day + 5 of monitoring indicating that they may have experienced improved food/water intake and/or kidney function during adenine induced interstitial nephritis.
Polyuria and polydipsia are the most common and earliest clinical manifestations of CKD. Adenine treated animals increased their urine output and water intake in association with impaired kidney function after adenine treatment. However, GA experienced a significant improvement in this marker of kidney function compared to WA at day 28. Anti-diuretic hormone (ADH) assists in controlling the body's water and electrolyte balance by influencing the water excretion by the kidneys 30 . When blood osmolality increases, ADH secretion is stimulated and causes the insertion of water channels into cells lining the collecting ducts, allowing water reabsorption to occur. Tubular reabsorption of sodium is dependent on sympathetic tone and angiotensin II acting via the effects of ADH and aldosterone to conserve water and sodium 31 . Decreased urine output in GA suggests improved reabsorption of water in GA and that Gunn rat kidneys may be able to respond to ADH secretion by increasing water reabsorption in the collecting tubule. This hypothesis agrees with published data indicating that exogenous bilirubin (10 μ M) significantly reduces urine production and urinary excretion of sodium, suggesting improvement in kidney vascular resistance and tubular function in an ischemia-isolated, perfused rat kidney ex vivo 18 . Impaired kidney function is reflected by the kidneys' tubules ability to maintain plasma urea and creatinine clearance 32 . Kidney dysfunction was manifested by a significant elevation of serum urea and creatinine concentrations in adenine-treated animals at day 0 versus baseline (−10). However, the concentrations of these analytes improved similarly during the monitoring period from day 14 and 28. Adenine-treated animals showed a clear progressive increment in urinary excretion of urea and creatinine; however, these effects plateaued earlier in Gunn rats and were significantly reduced in Gunn rats at day 14 and 28. Our results showed increased proteinuria and phosphate excretion in GA compared to WA at day 0; however, this effect was transient and was significantly reduced compared to Wistar animals at day 14 and 28, respectively. Proposed mechanisms which induce proteinuria include oxidative stress, inflammation and initiation and progression of tubulo-interstitial fibrosis 33 . A recent clinical study demonstrated that IgA nephropathy patients with mildly elevated serum bilirubin concentrations had reduced urinary protein concentration, serum creatinine and improved eGFR 8 . In addition, serum bilirubin concentrations were inversely associated urinary albumin excretion in hypertensive patients 34 and 24 hour urinary protein excretion in both diabetic and non-diabetic adults 35 . These data suggest that bilirubin could protect from glomerular injury, or improve tubular protein reabsorption after kidney inflammation had been induced. However, our data do not fully support these conclusions and are likely a consequence of the model used in this investigation which induced tubulo-interstitial rather than glomerular disease.
Chronic kidney disease is accompanied by characteristic abnormalities in lipid metabolism, which is reflected by elevated plasma lipid levels in patients with all stages of CKD 36 . It is important to note that Gunn rats (control or treated with adenine) possessed reduced total cholesterol concentrations compared to littermate controls, in agreement with previous findings 37,38 . Gunn rats maintained a hypocholesterolemic state throughout kidney disease induction which might indicate the importance of bilirubin induced hypocholesterolaemia, on protection from vascular disease in patients with CKD.
Dihydroxyadenine crystal deposition induces degenerative changes in the kidney tubules and causes kidney dysfunction with decreased concentrations of serum calcium and increased concentrations of phosphate 24,28,39 . Although serum calcium concentrations did not change in adenine-treated animals during the experimental period; a significant increase in urinary excretion of calcium was observed on day 14 and 28 in both groups. These results suggest dysfunctional calcium reabsorption and/or up-regulation of bone reabsorption leading to calcium excretion 40 . Although serum phosphate concentrations were elevated in GA at day 0 and normalised thereafter, reabsorption of filtered phosphorus (inferred from urinary phosphate output) was significantly improved in GA when compared to WA at day 28. Increased serum phosphate and calcium concentrations lead to vascular calcification which is an important risk factor for CKD in hemodialysis patients, and is associated with increased risk of atherosclerosis, ischemic heart disease, and vascular stiffening 40 . Previous studies have demonstrated an inverse relationship between bilirubin concentrations and vascular calcification 41,42 . Although our results do not support these findings, it is possible that mildly elevated bilirubin may protect from DHA-induced oxidative stress in a clinically relevant condition such as APRT deficiency.
Despite the lack of significant differences in kidney function parameters (i.e. serum urea and creatinine concentrations) between the Wistar controls and Gunn rats-treated with adenine, potentially important differences in antioxidant defences and oxidative stress biomarkers were noted between the two groups. As expected, Gunn rats possessed significantly elevated serum total bilirubin concentrations throughout the experimental period. It is presumed that the protective effects of bilirubin, shown in clinical studies, on kidney dysfunction are due to bilirubin's antioxidant properties 18,19,21 . Importantly, the concentrations of GSH and total reduced thiols were elevated (albeit not significantly so) in GA compared to WA at day 0 and 14.
These data are in agreement with previous studies including individuals with benign hyperbilirubinemia who also possess significantly elevated GSH and SH concentrations compared to controls, the concentration of which was positively correlated with unconjugated bilirubin (UCB) concentrations 37,43 . The glutathione antioxidant system is regarded as an important marker of oxidative stress in chronic kidney disease 44,45 . Previous studies have shown that the uraemic state is associated with low concentrations of GSH and reduced thiols (SH), which continue to decrease with the progression of kidney failure in dialysis patients 45 . Our findings, therefore, may indicate that bilirubin plays an important role in maintaining circulating thiol status providing an important pool of antioxidant capacity, which can protect proteins and lipids from oxidation by oxidants produced during inflammation, namely hypochlorous acid produced from myeloperoxidase (MPO) 46 . The importance of MPO-catalysed protein and lipid adduct formation has been considered an important mechanistic link between oxidation, atherosclerosis and kidney disease 47 , which bilirubin effectively inhibits 46 . A positive relationship between thiol oxidation, protein carbonylation and kidney dysfunction was reported in CKD patients who underwent dialysis 48 . Moreover, dialysis patients have greater plasma MPO activities and concentrations 49 in addition to increased protein carbonyl concentrations 50 , which are stable biomarkers of protein oxidation and damage 44 . Individuals with elevated bilirubin concentrations have decreased protein carbonyl concentrations 37 . Therefore, to determine whether elevated bilirubin and thiol/glutathione concentrations could protect against systemic oxidative stress in this model we measured systemic protein carbonyl concentrations. In the present study, GA demonstrated significantly decreased concentrations of protein carbonyls at all-time points compared to WA. Concomitantly, GA showed significantly decreased serum F 2 -isoprostane concentrations, the gold standard biomarker of lipid peroxidation that is associated with inflammation and oxidant stress 51 . These results support the protective effect of bilirubin against protein oxidation and lipid peroxidation in ex vivo 52 and in vivo 53 studies. The effect of bilirubin could be due to direct chloramine quenching 46 , or its thiol preserving capacity, either of which could protect proteins and lipids from MPO-induced oxidation.
In this study, we also report the deposition of bright symmetric crystalline DHA structures in the tubular lumen of adenine-treated animals. These deposits stimulate a local inflammatory response (mostly lymphocyte) and giant cell infiltration which were detected 28 days after adenine administration ceased. Macrophage infiltration triggers the initiation of interstitial fibrosis which results in collagen deposition in the interstitial space 27,54 . Lack of clear differences in histological grading (eg. inflammation, calcification and fibrosis) between the adenine-treated groups indicates that elevated bilirubin does not protect the tissue per se, from inflammation, calcification and oxidative injury. In contrast to plasma levels of F 2 -isoprostanes, kidney F 2 -isoprostane concentrations were not significantly different between the groups. Furthermore, the renal excretion of the stable DNA oxidation biomarker 8-oxo-7,8-dihydro -2′ -deoxyguanosine (8-oxodG), when expressed relative to urinary creatinine concentrations, was not different between the groups. Urinary 8-oxodG is considered to reflect global oxidation of the nucleotide pool, which is largely intracellular, and therefore acts as a whole-body marker of oxidative stress 55 . With contrasting improvements in oxidative damage within the vascular and kidney/organ compartments, these data indicate that bilirubin's protective effects are focussed within the circulatory compartment where it is bound to albumin, as supported by pharmacokinetic and volume of distribution calculations 56  Despite some potentially important findings reported here, it should be noted that this study has several limitations. Administration of adenine sulphate suspension contributed to variability in the pathophysiological sequelae of administration and therefore, our ability to detect statistically significant differences between the groups. Longer-term adenine administration (e.g. < 4 weeks) modulates kidney function with less reversibility 25 and therefore could provide more reliable and thus significant outcomes. We did not measure blood pressure and kidney injury biomarkers including kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin and therefore, we could not determine whether bilirubin's antioxidant effects, lead to improvements in vascular tone and reactivity.
In conclusion, we have demonstrated that Gunn rats maintain body weight, experience reduced polyuria and polydipsia when compared to Wistar rats after treatment with adenine. Elevated circulating bilirubin concentrations prevented protein and lipid oxidation within the circulatory compartment during kidney inflammation in Gunn rats. However, these data suggest that endogenously elevated bilirubin does not protect from kidney dysfunction (as assessed by accumulation of urea/creatinine) or inflammation, fibrosis/organ oxidative damage. This study suggests that endogenously elevated bilirubin may improve red-ox status and protect from kidney injury-associated vascular damage. Bilirubin protects from cardiovascular events in patients undergoing chronic hemodialysis 3,17 . Although bilirubin might not protect from the decrement of kidney function over time, it might protect from endothelial dysfunction, vascular calcification, hypertension, lipoprotein oxidation and dyslipidemia, which are strongly implicated with CKD and contribute to longer term cardiovascular mortality 10 .

Animals. Breeding pairs of heterozygote (genotyped) Gunn rats were purchased from the Rat
Research and Resource Center (Columbia, MO, USA). Rats were housed at Griffith University (12-h light: dark cycle, constant temperature (22 °C) and humidity (60%) and had continuous access to standard laboratory food pellets (Speciality Feeds, Glen Forrest, Australia) and fresh water. Male homozygous Gunn rat offspring possess jaundice at birth, were ear-tagged, and housed together with male littermate (non-jaundiced) controls after weaning. Ten week-old male Gunn and Wistar rats were used in this experiment and all procedures were performed in accordance with relevant guidelines and approved by the Griffith University Animal Ethics Research Committee prior to commencement of experimentation (MSC/12/12).
Oral adenine model. Animals were randomly divided into four groups. Gunn (GA) and littermate Wistar (WA) rats (n = 12 per group) were orally administered adenine in a suspension in 0.5% methycellulose (300 mg adenine sulphate/kg body weight; Sigma-Aldrich, Australia) once daily for 10 days (i.e. induction period) and monitored for 28 days thereafter as tubulo-interstitial nephritis developed 24,28,29 . Control Gunn (GC; n = 9) and littermate Wistar rats (WC; n= 6) were given 0.5% methylcellulose (mL/ kg body weight) daily by oral gavage. Animal body weights were measured daily for 10 days during adenine treatment (induction) and fortnightly thereafter. Blood and urine samples were collected at baseline (− 10 days), after induction (day 0) and then fortnightly (days 14 and 28) for biochemical assessment of kidney function. Animals were housed in metabolic cages for 24 hours with free access to food and water. Twenty-four hour urine output/composition and water consumption was collected at day − 10, 0, 14 and 28. Urine excretion and water intake volume were measured manually. Approximately 1 mL of blood was collected from the tail tip with brief isoflurane anaesthesia (3% in 100% O 2 ; 1-2 L min −1 ) and transferred into serum and EDTA vacutainers. On day 28, all animals were anaesthetised using an intraperitoneal injection of pentobarbital sodium (concentration 60 mg/mL; 100 μ L/100 g). A midline laparotomy/thoracotomy was performed, and approximately 5 mL of whole blood was collected using syringe from the thoracic cavity and transferred into serum and EDTA vacutainers. Animals were euthanized by removing the heart. Kidneys were also harvested for histological examination. Kidney tissue was frozen in liquid nitrogen and then crushed using a mortar and pestle and kept at − 80 °C for F 2 -isoprostane analysis.
Sample preparation. Whole blood was centrifuged (Thermo Scientific 5810R, Australia) at 2500 × g for 10 min (4 °C). Serum/plasma and urine aliquots were prepared immediately and stored at − 80 °C until analysis. Trichloroacetic acid (10%; ChemSupply, Australia) was added to serum aliquots in 1:1 ratio, which were then vortexed and centrifuged at 2500 × g for 5 min. Supernatant was stored at − 80 °C for reduced and oxidized glutathione analysis.
Biochemistry. Serum samples were analysed for creatinine, uric acid, urea, calcium, phosphate and albumin using a COBAS Integra 400 blood chemistry analyser (Roche Diagnostics, Australia) to monitor the extent of kidney dysfunction. Systemic markers of liver function (alanine aminotransferase, aspartate aminotransferase, γ -glutamyltransferase activity), glucose, lipid parameters (total cholesterol and triglyceride), lipase activity, total protein, total and direct (conjugated) bilirubin were assessed using the same analyser. Cholesterol analyses were conducted using appropriate lipid standards (Calibrator for Automated Systems Lipids) and quality controls (Precinorm Control Clin Chem Multi 1 and 2; Roche Diagnostics, Australia).
Kidney excretion of creatinine, urea, calcium and phosphate were also analysed in urine samples. Total output of the solutes above was calculated from the volume of urine excreted and the concentration of each analyte within each sample. Total protein in urine was quantified using appropriate standards and quality controls (total protein urine quality control; Roche Diagnostics, Australia). Ionic composition of urine (e.g. chloride, potassium and sodium) was assessed using ion-selective electrodes, in addition to calibrators including direct and reference electrolyte solutions (Roche Diagnostics, Australia). All the tests were conducted in duplicate.

Measurement of reduced and oxidised glutathione concentrations. Reduced (GSH) and oxi-
dised (GSSG) glutathione concentrations were quantified in trichloroacetic acid supernatants of serum using N-ethylmaleimide (NEM) and o-phthalaldehyde (OPA) via a modified method of Hissin and Hilf 57 . The fluorescence intensity of GSSG and GSH were determined using black 96 well plates (Corning, Australia) at 350 nm (excitation) and 420 nm (emission) with a Fluoroskan Ascent 96 well plate reader (Thermo Scientific, Australia). The concentrations of GSH and GSSG in the samples were determined in triplicate using external standards ranging from 1.56-50 μ M. The co-efficient of variation for this method was 3.4%.

Measurement of reduced thiol concentrations.
Reduced thiol concentrations were measured using 5,5-dithiobis(2-nitrobenzoic acid) (DTNB; Sigma-Aldrich, Australia) reagent according to the method of Hawkins et al. 58 . Reduced thiols react with DTNB to generate 5-thio-2-nitrobenzoic acid (TNB), which was quantified in triplicate at 415 nm using a 96-well plate reader (Multiskan FC, Thermo Scientific, Australia). Reduced glutathione (0-0.5 mM) served as an external standard and thiol concentrations were expressed initially in μ M and then converted to nmol/mg of protein. The co-efficient of variation for this method was 2.6%.

Measurement of protein carbonyl concentrations.
Detection of protein carbonyl with 2,4-dinitrophenylhydrazine in EDTA plasma was performed using enzyme-linked immunoassay (ELISA) kits (Sapphire Biosciences, Australia) with the above-mentioned 96-well plate reader. Results were expressed in nmols/mg of protein and all analyses were conducted in duplicate. The co-efficient of variation for this assay was 6.8%.

Measurement of F 2 -isoprostanes.
Samples were analysed in duplicate using our previously published method 59 . Total F 2 -isoprostanes were extracted from plasma and kidney tissue after saponification with methanolic NaOH. Samples were spiked with 8-iso-PGF2α -d4 (Cayman Chemicals, USA) as an internal standard and incubated at 42 °C for 60 min. Samples were then acidified (pH 3) with hydrochloric acid, and hexane added and samples, mixed for 10 min before centrifugation. The supernatant was removed and the remaining solution extracted with ethyl acetate and dried under nitrogen. Samples were reconstituted with acetonitrile, transferred into vials with silanised glass inserts and dried. Derivatisation with pentafluorobenzylbromide and diisopropylethylamine and incubation at room temperature for 30 min followed. Samples were then dried under nitrogen before pyridine, Bis(trimethylsilyl)trifluoroacetamide (99%) and trimethylchlorosilane (1%) were added and incubated at 45 °C for 20 min. Finally, hexane was added samples were mixed, then 1μ L was injected for analysis using gas chromatography tandem mass spectrometry (Varian, Australia) in negative chemical ionization mode. The coefficient of variation for this assay was 4.5%.
Measurement of urine 8-oxo-7,8-dihydro-2′-deoxyguanosine. Each sample (urine samples, pooled urine and controls) at − 80 °C was thawed to room temperature and analysed for 8-oxo-7 ,8-dihydro-2′ -deoxyguanosine (8-oxodG) according to our previously published method 60 . Samples were vigorously mixed, sonicated for one minute, and then centrifuged at 15,000 × g for 10 min. A 50-μ L clear supernatant of each sample was directly injected into the HPLC-ESI/MS/MS system. HPLC separations were performed using an Agilent HPLC 1200 pump (G1311A and micro vacuum degasser, G1322A), equipped with a thermostatted (set at 4 °C) well-plate autosampler (G1329A), linked to a 3200 QTAP mass spectrometer (from Applied Biosystems/MDS Sciex). Only the eluate fraction of 8-oxodG was delivered into the spectrometer, others were diverted to waste. All data were acquired and processed by Analyst ® Software 1.4.2 (MDS Inc., Concord, ON, Canada). Creatinine concentrations in diluted urine were measured at 505 nm by a Roche/Hitachi 902 Analyzer (Roche Diagnostics GmbH, Mannheim, Germany) using the Jaffe method with rate-blanked and compensated. Urine 8-oxo-7,8-dihydro-2′ -deoxyguanosine results were expressed relative to creatinine concentrations. The coefficient of variation for this assay was 4.5%.
Histological studies. Harvested kidneys were dissected into multiple sagittal and transverse sections for histological analysis. Sections were fixed in 10% formaldehyde in phosphate-buffered saline and embedded in paraffin. Paraffin sections were stained with haematoxylin-eosin (HE) for the presence of DHA crystals, calcification, inflammatory cells (including foreign body giant cells) and Masson's Trichrome for fibrosis. The histological kidney sections were examined in a blinded fashion by the author (AKL) using light microscope (Olympus BX43, Notting Hill, Victoria, Australia) under total 100× magnification power with a polarizer for detection of DHA crystals. The severity of each morphological Scientific RepoRts | 5:15482 | DOi: 10.1038/srep15482 features were graded from 0 to + + + and the histological scores for adenine-treated animals were assessed in a semi-quantitated fashion (see statistical analysis). Statistical analysis. Statistical analysis was performed using SigmaPlot software (version 11.0). All values are expressed as mean ± standard deviation. All data were tested for normality and equality of variance, with appropriate parametric or non-parametric statistical tests applied. Two-tailed, unpaired t-tests (Student's t test) or Rank Sum tests were applied for pairwise comparisons. Two way repeated-measures ANOVA was used to compare the differences between groups over time. Bonferroni post-hoc tests were applied to determine at which time-points significant differences between groups occurred. The Fisher exact test was used to determine differences in histological grading between Gunn and Wistar animals. A P < 0.05 was considered statistically significant.